CN112213498A - D-dimer determination kit and application thereof - Google Patents
D-dimer determination kit and application thereof Download PDFInfo
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- CN112213498A CN112213498A CN202011055793.9A CN202011055793A CN112213498A CN 112213498 A CN112213498 A CN 112213498A CN 202011055793 A CN202011055793 A CN 202011055793A CN 112213498 A CN112213498 A CN 112213498A
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- 229960002684 aminocaproic acid Drugs 0.000 claims abstract description 35
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 25
- 238000003149 assay kit Methods 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 12
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- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention provides a D-dimer assay kit and application thereof, relates to the technical field of detection, and provides the D-dimer assay kit, wherein mouse anti-human D-dimer monoclonal antibody latex particles are prepared by combining a mouse anti-human D-dimer monoclonal antibody with 6-aminocaproic acid modified polystyrene latex particles, and the 6-aminocaproic acid is modified on the surfaces of the polystyrene latex particles, so that the chain length of carboxyl groups on the surfaces of the polystyrene latex particles can be effectively prolonged. Meanwhile, the invention can further improve the combination efficiency of the mouse anti-human D-dimer monoclonal antibody and the 6-aminocaproic acid modified polystyrene latex particles by limiting the pH value of the PBST solution for dissolving the 6-aminocaproic acid to be 8.0-9.0. In addition, the kit also has the advantages of simple operation method, rapidness, accuracy, high sensitivity, strong specificity, good stability, normal-temperature storage and the like.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a D-dimer determination kit and application thereof.
Background
The fibrinolytic system is the most important anticoagulant system in human body, and has important function for keeping permeability of human vascular wall and exchanging substance. Hitherto, for functional detection of fibrinolysis system, the immune turbidimetric method is mainly used to quantitatively detect various soluble fragments formed by degrading fibrinogen and cross-linked fibrin with plasmin, which are collectively called Fibrinogen Degradation Products (FDP), wherein D-dimer is the minimum fragment in the degradation products and is a specific degradation product of cross-linked fibrin.
In recent years, various valuable D-dimer detection methods have been established, mainly including latex agglutination, enzyme-linked immunosorbent assay, fluorescent antibody detection, immuno-gold labeling and latex immunoturbidimetry. The latex immunoturbidimetry is more suitable for emergency detection and is used for qualitative or semi-quantitative detection due to simple and rapid operation.
However, latex microsphere-antibody complexes have been prepared so far using coupling reagents for coupling and binding of antibodies to rigid microspheres tends to be influenced by thermodynamic factors, resulting in surface binding of antibodies affecting active site exposure.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
It is a primary object of the present invention to provide a D-dimer assay kit that alleviates at least one of the technical problems of the prior art.
The second purpose of the invention is to provide the application of the D-dimer determination kit in the detection of a hemagglutination analyzer and a biochemical analyzer.
The invention provides a D-dimer assay kit, which comprises a D-dimer R1 reagent and a D-dimer R2 reagent;
the D-dimer R1 reagent includes a buffer solution, a coagulant, and a preservative;
the D-dimer R2 reagent comprises mouse anti-human D-dimer monoclonal antibody latex particles, a buffer solution, a stabilizer and a preservative;
the mouse anti-human D-dimer monoclonal antibody latex particle is prepared by combining a mouse anti-human D-dimer monoclonal antibody with a 6-aminocaproic acid modified polystyrene latex particle;
the 6-aminocaproic acid modified polystyrene latex particles are prepared by the following method:
dissolving the pretreated polystyrene latex particles in PBST solution of 6-aminocaproic acid, and carrying out solid-liquid separation after reaction to obtain the polystyrene latex particles modified by the 6-aminocaproic acid;
wherein the pH of the PBST is 8.0-9.0.
Further, the pretreatment comprises the steps of adding a polystyrene latex particle aqueous solution into an MES solution, then sequentially adding an EDC solution and an NHS solution, mixing and reacting, and carrying out solid-liquid separation to obtain pretreated polystyrene latex particles;
preferably, an EDC solution is prepared with a 0.1M MES solution at a concentration of 100 mg/ml;
preferably, a 0.1M MES solution is used to prepare a NHS solution with a concentration of 100 mg/ml;
preferably, the MES solution has a pH of 6.0;
preferably, the concentration of the aqueous solution of polystyrene latex particles is 10 mg/ml.
Further, 400ul of an aqueous solution of polystyrene latex particles having a concentration of 10mg/ml was added to 5ml of 0.1M MES solution, then 100ul of EDC solution and 72ul of NHS solution were sequentially added thereto, and stirred at room temperature for 2 hours; after the reaction is finished, centrifugal washing is carried out at 3500 rpm; finally, 5ml of PBST solution of 6-aminocaproic acid with the concentration of 100mM/L is used for redissolving, and then the mixture is stirred at room temperature overnight; after the reaction, the latex was centrifuged at 12000rpm and finally dispersed in 5ml of 0.1M MES solution to obtain the 6-aminocaproic acid-modified polystyrene latex particles.
Further, adding the mouse anti-human D-dimer monoclonal antibody into the PBST solution of the activated 6-aminocaproic acid modified polystyrene latex particles to obtain mouse anti-human D-dimer monoclonal antibody latex particles;
the pH of the PBST is 7.0-7.4.
Further, adding 6-aminocaproic acid modified polystyrene latex particles into 100ul of EDC solution, then adding 72ul of NHS solution, stirring for 2 hours at room temperature, activating, centrifugally washing at 12000rpm after the reaction is finished, dispersing in 2ml of PBST, then diluting 26ul of mouse anti-human D-dimer monoclonal antibody with 2ml of PBST, and then dropwise adding into the microsphere solution to obtain the mouse anti-human D-dimer monoclonal antibody latex particles.
Further, the buffer solution in the D-dimer R1 reagent comprises PBS buffer solution;
preferably, the procoagulant in the D-dimer R1 reagent comprises PEG 8000.
Further, the buffer solution in the D-dimer R2 reagent comprises a PBS buffer solution, preferably a PBS buffer solution with pH of 7.4;
preferably, the stabilizing agent in the D-dimer R2 reagent comprises 0.3 wt% BSA, 100mM glycine, and 0.3 wt% tween 40;
preferably, the preservatives in the D-dimer R1 reagent and the D-dimer R2 reagent include Proclin-300;
preferably, the concentration of the latex particles of the mouse anti-human D-dimer monoclonal antibody in the D-dimer R2 reagent is 2-6 mg/mL.
Further, the polystyrene latex particle is a latex in a core-shell form, the latex core is a styrene polymer, and the latex shell is a copolymer formed by styrene, n-butyl acrylate and methacrylic acid.
Further, the particle size of the milk core is 120nm, the average particle size of the 6-aminocaproic acid modified polystyrene latex particles is 180nm-220nm, and the weight ratio of styrene to n-butyl acrylate in the milk shell is 1: 1, the weight of the milk shell is 10-30% of the total weight of the polystyrene latex particles, and the weight of the methacrylic acid is 1.0-5.0% of the total weight of the latex particles.
In addition, the invention also provides application of the D-dimer determination kit in detection of a hemagglutination analyzer and a biochemical analyzer.
Compared with the prior art, the invention has the following beneficial effects:
according to the D-dimer assay kit provided by the invention, the mouse anti-human D-dimer monoclonal antibody latex particles are prepared by combining the mouse anti-human D-dimer monoclonal antibody with the 6-aminocaproic acid modified polystyrene latex particles, and the chain length of carboxyl groups on the surfaces of the polystyrene latex particles can be effectively prolonged by modifying the 6-aminocaproic acid on the surfaces of the polystyrene latex particles. Meanwhile, the pH value of the PBST solution for dissolving the 6-aminocaproic acid is limited to be 8.0-9.0, so that the combination efficiency of the mouse anti-human D-Dimer monoclonal antibody and the 6-aminocaproic acid modified polystyrene latex particles can be further improved, the latex particles are modified by the antibody with high activity on the basis of effectively saving the cost, and the D-Dimer in-vitro diagnostic kit with high sensitivity and high activity is prepared. In addition, the kit also has the advantages of simple operation method, rapidness, accuracy, high sensitivity, strong specificity, good stability, normal-temperature storage and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1a is a graph showing the results of particle size distribution and PDI value of blank microspheres obtained in Experimental example 2;
FIG. 1b is a graph showing the results of surface potential of blank microspheres according to Experimental example 2 of the present invention;
FIG. 2 is a graph showing the results of particle size distribution and PDI value of 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method of comparative example 1, provided in Experimental example 2 of the present invention;
FIG. 3a is a graph showing the results of particle size distribution and PDI value of 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method provided in application example 1, according to Experimental example 2 of the present invention;
FIG. 3b is a graph showing the results of surface potential of 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method provided in application example 1, according to Experimental example 2 of the present invention;
FIG. 4a is a graph showing the results of particle size distribution and PDI value of 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method provided in application example 2 of the present invention, provided in Experimental example 2;
FIG. 4b is a graph showing the results of surface potential of 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method provided in application example 2, according to Experimental example 2 of the present invention;
FIG. 5a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 1;
FIG. 5b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 1 according to Experimental example 2;
FIG. 6a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 3 according to Experimental example 2;
FIG. 6b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 3 according to Experimental example 2;
FIG. 7a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 4 according to Experimental example 2;
FIG. 7b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 4 according to Experimental example 2;
FIG. 8a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 5;
FIG. 8b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 5 according to Experimental example 2;
FIG. 9a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 6 according to Experimental example 2;
FIG. 9b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 6 according to Experimental example 2;
FIG. 10a is a graph showing the results of particle size distribution and PDI value of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 7;
FIG. 10b is a graph showing the results of surface potential of mouse anti-human D-dimer monoclonal antibody latex particles prepared by the method of example 7 according to Experimental example 2;
FIG. 11 is a standard curve provided in Experimental example 1 of the present invention.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
In general, unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods that are well known in the art, and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the present invention, there is provided a D-dimer assay kit comprising a D-dimer R1 reagent and a D-dimer R2 reagent;
the D-dimer R1 reagent includes a buffer solution, a coagulant, and a preservative;
the D-dimer R2 reagent comprises mouse anti-human D-dimer monoclonal antibody latex particles, a buffer solution, a stabilizer and a preservative;
the mouse anti-human D-dimer monoclonal antibody latex particle is prepared by combining a mouse anti-human D-dimer monoclonal antibody with a 6-aminocaproic acid modified polystyrene latex particle;
the 6-aminocaproic acid modified polystyrene latex particles are prepared by the following method:
dissolving the pretreated polystyrene latex particles in PBST solution of 6-aminocaproic acid, and carrying out solid-liquid separation after reaction to obtain the polystyrene latex particles modified by the 6-aminocaproic acid;
wherein the pH of the PBST is 8.0-9.0.
According to the D-dimer determination kit provided by the invention, the chain length of carboxyl groups on the surfaces of the polystyrene latex particles can be effectively prolonged by modifying 6-aminocaproic acid on the surfaces of the polystyrene latex particles. Meanwhile, the pH value of the PBST solution for dissolving the 6-aminocaproic acid is limited to be 8.0-9.0, so that the combination efficiency of a mouse anti-human D-Dimer monoclonal antibody and the 6-aminocaproic acid modified polystyrene latex particles can be further improved, the latex particles are modified by the antibody with high activity on the basis of effectively saving the cost, and the D-Dimer in-vitro diagnostic kit with high sensitivity and high activity is prepared. In addition, the kit also has the advantages of simple operation method, rapidness, accuracy, high sensitivity, strong specificity, good stability, normal-temperature storage and the like.
The pH of PBST may be, but is not limited to, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0, for example.
In some preferred embodiments, the pretreatment comprises adding an aqueous solution of polystyrene latex particles into a MES (2- (N-morpholine) ethanesulfonic acid) solution, then sequentially adding an EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) solution and an NHS (N-hydroxysuccinimide) solution, mixing, reacting, and carrying out solid-liquid separation to obtain the pretreated polystyrene latex particles.
The combination efficiency of the polystyrene latex particles and the mouse anti-human D-dimer monoclonal antibody in the subsequent steps can be further improved by pretreating the EDC solution and the NHS solution.
Preferably, preparing EDC solution with the concentration of 100mg/ml by using MES solution;
preferably, an NHS solution is prepared with a MES solution at a concentration of 100 mg/ml;
preferably, the MES solution has a pH of 6.0 and a concentration of 0.1M;
preferably, the concentration of the aqueous solution of polystyrene latex particles is 10 mg/ml.
In some preferred embodiments, a murine anti-human D-dimer monoclonal antibody is added to a PBST solution of activated 6-aminocaproic acid modified polystyrene latex particles to obtain murine anti-human D-dimer monoclonal antibody latex particles;
the pH of the PBST is 7.0-7.4, and can be, for example, but not limited to, 7.0, 7.1, 7.2, 7.3, or 7.4.
The combination efficiency of the D-dimer monoclonal antibody and the polystyrene latex particles can be further improved by regulating and controlling the pH value of a PBST solution of the reaction of the mouse anti-human D-dimer monoclonal antibody and the polystyrene latex particles modified by 6-aminocaproic acid.
In some preferred embodiments, the stabilizing agent in the D-dimer R2 reagent comprises 0.3% BSA, glycine, and 0.3% tween 40.
The stabilizer in the D-dimer R2 reagent prepared by the specific proportion and the raw materials has a better stabilizing effect, can effectively prolong the storage time of the kit, and saves the cost.
Based on the fact that the D-dimer in the sample to be detected and the latex particles of the mouse anti-human D-dimer monoclonal antibody in the D-dimer determination kit provided by the invention can generate antigen-antibody agglutination reaction, the agglutination is generated, the turbidity of a reaction system is increased, the light transmittance is reduced, the change rate of the turbidity is in direct proportion to the concentration of the D-dimer in the sample, and the content of the D-dimer in the sample can be calculated by determining the change rate of the light transmittance by using a hemagglutination analyzer or a biochemical analyzer. Based on the above, the second aspect of the invention also provides the application of the D-dimer assay kit in the detection of a hemagglutination analyzer and a biochemical analyzer.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The main reagent information used in the examples of the present invention is as follows:
edc.hcl (analytical pure, TCI), NHS (analytical pure, TCI), MES (analytical pure, alatin), PEG-8000 (analytical pure, TCI), Proclin-300 (analytical pure, Sigma), bovine serum albumin BSA (bio-reagent, solibao), HEPS (analytical pure, alatin), potassium chloride (analytical pure, alatin), glycine (bio-reagent, alatin), D-dimer calibrator (sun).
EXAMPLE 1 preparation of D-dimer assay kit
1. Preparation of D-dimer R1 reagent:
adding PEG8000(5 wt%) and Proclin-300(0.005 wt%) into PBS buffer solution, stirring and mixing uniformly to obtain D-dimer R1 reagent;
2. preparation of D-dimer R2 reagent:
(1) preparation of 6-aminocaproic acid modified polystyrene latex particles
To 5ml of a 0.1M MES (pH 6.0) solution, 400ul of an aqueous solution of polystyrene latex particles having a concentration of 10mg/ml was added, followed by 100ul of EDC and 72ul of NHS solution, and the mixture was stirred at room temperature for 2 hours. After the reaction, 3500rpm centrifugal washing was performed. Finally, the mixture was reconstituted with 5ml of 6-aminocaproic acid (dissolved in PBST) at a concentration of 100mM/L, stirred overnight at room temperature, centrifuged at 12000rpm after the reaction was complete, and finally dispersed in 5ml of a 0.1M MES solution.
Here, EDC solution and NHS solution at a concentration of 100mg/ml were prepared using MES (pH 6.0) solution of 0.1M.
In this step, the pH of the PBST was 8.0.
(2) Binding of antibody latex
The sample was added to 100ul EDC, then 72ul NHS solution was added, stirred at room temperature for 2 hours for activation, after the reaction was completed, centrifuged at 12000rpm and washed and dispersed in 2ml PBST, then 26ul mouse anti-human D-dimer antibody was diluted with 2ml PBST, then added dropwise to the microsphere solution, and finally the latex was resuspended in PBS buffer containing stabilizers (0.3% BSA, glycine and 0.3% Tween 40) and Proclin-300.
In this step, the pH of the PBST was 7.0.
Example 2
This example differs from example 1 in that in step (1), the pH of the PBST was 9.0.
Example 3
This example differs from example 1 in that in step (2), the pH of the PBST was 7.2.
Example 4
This example differs from example 1 in that in step (2), the pH of the PBST was 7.4.
Example 5
This example differs from example 2 in that in step (2), the pH of the PBST was 7.0.
Example 6
This example differs from example 2 in that in step (2), the pH of the PBST was 7.2.
Example 7
This example differs from example 2 in that in step (2), the pH of the PBST was 7.4.
Comparative example 1
This comparative example differs from example 1 in that in step (1), the pH of the PBST was 7.4.
Experimental example 1 method for detecting sample with kit
The purchased D-dimer calibrator was dissolved in 0.5ml of distilled water and prepared into 6 calibrator solutions of different concentrations, 6.080, 3.040, 1.520, 0.760, 0.380 and 0.190ug/ml, respectively, with physiological saline. Taking the operation of the eastern Asia CA550 hemagglutination apparatus in Japan as an example: measuring the wavelength of 575nm, respectively removing calibrator solutions (20ul) with different concentrations, adding D-dimer diluent (5ul), reacting at 37 ℃ for 30 seconds, adding D-dimer R1(100ul), reacting for 60 seconds, adding D-dimer R2(100ul), measuring the absorbance values of the 10 th and 90 th seconds, calculating the absorbance difference, repeatedly measuring for 3 times in each tube, taking the average value of the absorbance difference measured for 3 times in each calibration tube as the ordinate, taking the corresponding concentration as the abscissa, and making a 'concentration-absorbance difference' standard curve: y 0.20317 x-0.01956, R20.99981 (as shown in fig. 11). And taking a sample to be detected, measuring the absorbance difference value of the sample by the same method, and substituting the absorbance difference value into the standard curve to calculate the content of the D-dimer in the sample to be detected. If the concentration of the D-dimer in the sample is beyond the range of the calibration curve, the sample needs to be diluted properly and then detected in order to ensure the detection accuracy.
Experimental example 2 evaluation of analytical Properties of assay kit
1. Surface potential and particle size variation
First, the particle size distribution and surface potential (Malvern Zetasizer Nano ZS) of the purchased 0.12um microspheres were examined at 130nm and-55 mV, respectively, as shown in FIGS. 1a and 1 b.
(1) The particle size and dispersibility of the 6-aminocaproic acid-modified polystyrene latex particles prepared by the preparation method provided in comparative example 1 are shown in FIG. 2, and the dispersibility is poor. And the Zeta number was determined to be close to 0, indicating that the dendron 6-aminocaproic acid was not available at a pH of 7.4.
(2) 6-aminocaproic acid-modified polystyrene latex particles were prepared using the preparation method provided in example 1, and their particle size, dispersibility, and surface potential were as shown in FIGS. 3a and 3 b. Zeta values close to-33 mV were determined, indicating that the branched 6-aminocaproic acid was available at a pH of 8.0.
(3) 6-aminocaproic acid-modified polystyrene latex particles were prepared using the preparation method provided in example 2, and their particle size and dispersibility were as shown in FIGS. 4a and 4 b. Zeta values close to-33 mV were determined, indicating that the dendrimer 6-aminocaproic acid was effective at a pH of 9.0.
(4) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 1 are shown in FIGS. 5a and 5b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
(5) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 3 are shown in fig. 6a and 6b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
(6) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 4 are shown in fig. 7a and 7b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
(7) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 5 are shown in fig. 8a and 8b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
(8) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 6 are shown in fig. 9a and 9b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
(9) The particle size, dispersibility and surface potential of the latex particles of the murine anti-human D-dimer monoclonal antibody prepared by the preparation method provided in example 7 are shown in fig. 10a and 10b, where the left side of a is the hydrated particle size and the right side results are the dispersion coefficient PDI values.
As can be seen from the above experiments, when the pH is 9.0 and 8.0 in step (1) and the antibody-binding pH is 7.4 in step (2), the dispersibility, particle size and the like are optimum.
2. Repeatability of
The D-dimer assay kit prepared by the preparation method provided by the embodiment 7 of the invention is used for carrying out repeatability investigation on D-dimer standard products with sun company identification values of 0.506ug/mL and 3.320ug/mL respectively, and the results are shown in Table 1, wherein the relative deviation is 0.9% and 0.5% respectively.
TABLE 1 sample repeatability
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A D-dimer assay kit, comprising a D-dimer R1 reagent and a D-dimer R2 reagent;
the D-dimer R1 reagent includes a buffer solution, a coagulant, and a preservative;
the D-dimer R2 reagent comprises mouse anti-human D-dimer monoclonal antibody latex particles, a buffer solution, a stabilizer and a preservative;
the mouse anti-human D-dimer monoclonal antibody latex particle is prepared by combining a mouse anti-human D-dimer monoclonal antibody with a 6-aminocaproic acid modified polystyrene latex particle;
the 6-aminocaproic acid modified polystyrene latex particles are prepared by the following method:
dissolving the pretreated polystyrene latex particles in PBST solution of 6-aminocaproic acid, and carrying out solid-liquid separation after reaction to obtain the polystyrene latex particles modified by the 6-aminocaproic acid;
wherein the pH of the PBST is 8.0-9.0.
2. The D-dimer assay kit according to claim 1, wherein the pretreatment comprises adding an aqueous solution of polystyrene latex particles to an MES solution, then adding an EDC solution and an NHS solution in sequence, mixing and reacting, and performing solid-liquid separation to obtain pretreated polystyrene latex particles;
preferably, preparing EDC solution with the concentration of 100mg/ml by using MES solution;
preferably, an NHS solution is prepared with a MES solution at a concentration of 100 mg/ml;
preferably, the MES solution has a pH of 6.0 and a concentration of 0.1M;
preferably, the concentration of the aqueous solution of polystyrene latex particles is 10 mg/ml.
3. The D-dimer assay kit according to claim 2, wherein 400ul of an aqueous solution of polystyrene latex particles having a concentration of 10mg/ml is added to 5ml of 0.1M MES solution, and then 100ul of EDC solution and 72ul of NHS solution are sequentially added thereto, followed by stirring at room temperature for 2 hours; after the reaction is finished, centrifugal washing is carried out at 3500 rpm; finally, 5ml of PBST solution of 6-aminocaproic acid with the concentration of 100mM/l is used for redissolving, and then the mixture is stirred at room temperature overnight; after the reaction, the latex was centrifuged at 12000rpm and finally dispersed in 5ml of 0.1M MES solution to obtain the 6-aminocaproic acid-modified polystyrene latex particles.
4. The D-dimer assay kit according to any one of claims 1 to 3, wherein a mouse anti-human D-dimer monoclonal antibody is added to a PBST solution of activated 6-aminocaproic acid-modified polystyrene latex particles to obtain mouse anti-human D-dimer monoclonal antibody latex particles;
the pH of the PBST is 7.0-7.4.
5. The D-dimer assay kit according to claim 4, wherein the latex particles of the mouse anti-human D-dimer monoclonal antibody are prepared by adding 6-aminocaproic acid modified polystyrene latex particles to 100ul of EDC solution, adding 72ul of NHS solution, stirring at room temperature for 2 hours, activating, dispersing in 2ml of PBST after centrifugal washing at 12000rpm after the reaction is completed, diluting 26ul of the mouse anti-human D-dimer monoclonal antibody with 2ml of PBST, and adding dropwise to the microsphere solution.
6. The D-dimer assay kit of claim 1, wherein the buffer solution in the D-dimer R1 reagent comprises PBS buffer solution;
preferably, the procoagulant in the D-dimer R1 reagent comprises PEG 8000.
7. The D-dimer assay kit of claim 1, wherein the buffer solution in the D-dimer R2 reagent comprises a PBS buffer solution, preferably a PBS buffer solution with pH of 7.4;
preferably, the stabilizing agent in the D-dimer R2 reagent comprises 0.3 wt% BSA, 100mM glycine, and 0.3 wt% tween 40;
preferably, the preservatives in the D-dimer R1 reagent and the D-dimer R2 reagent include Proclin-300;
preferably, the concentration of the latex particles of the mouse anti-human D-dimer monoclonal antibody in the D-dimer R2 reagent is 2-6 mg/mL.
8. The D-dimer assay kit according to claim 1, wherein said polystyrene latex particles are a core-shell form of latex, the latex core is a styrene polymer, and the latex shell is a copolymer of styrene, n-butyl acrylate, and methacrylic acid.
9. The D-dimer assay kit according to claim 8, wherein the particle size of said milk core is 120nm, the average particle size of said 6-aminocaproic acid modified polystyrene latex particles is 180nm to 220nm, and the weight ratio of styrene to n-butyl acrylate in said milk shell is 1: 1, the weight of the milk shell is 10-30% of the total weight of the polystyrene latex particles, and the weight of the methacrylic acid is 1.0-5.0% of the total weight of the latex particles.
10. Use of the D-dimer assay kit of any one of claims 1-8 in the detection of hemagglutination and biochemical analyzers.
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